This proposal continues our researches into the mechanism by which the synthesis of one of the body's most important energy reserves, glycogen, is initiated. A protein which we found covalently bound to glycogen and which we call glycogenin has now been found in a glycogen-free state in muscle. It is autocatalytic glycoprotein (self-glucosylating protein, SGP) that builds a maltosaccharide (malto-octaose) on itself that in turn primes glycogen synthesis. In this continuation proposal we will study how the self-glycosylating protein is initially glycosylated. We have narrowed the possible routes to that of placing one or two glucose residues on the protein via the unique glucose-tyrosine bond that constitutes the linkage of glycogen to glycogenin. For this purpose we will clone the cDNA for glycogenin SGP in order to learn the size of the protein coded for and to obtain recombinant protein as a test substrate for the glycosylation studies. Among the mechanisms that may regulate SGP in its role in initiating glycogen synthesis is covalent modification by multi-site phosphorylation, while modulation of the already activated SGP may occur via ATP and the UDPglucose substrate, the binding sites for which will be determined. Another regulatory phenomenon is the autoactivation of SGP that occurs when muscle is minced, simultaneously with that of a seemingly related structurally similar protein having Mr 42KDa (P42). When autoactivation is allowed to occur in presence of an alpha-glucosidase inhibitor SGP and P42 are replaced by a new family of higher mol. wt. glucosylated proteins. We will examine the structures and inter-relation of these additional proteins. Glycogenin (and SGP) represent control points of great potential importance in regulating glycogen synthesis. The dramatic effect of insulin in laying down glycogen may be exerted, in part, at this point. Without SGP, or active SGP, there would be no glycogen so that the sophisticated, endocrine controls and second messenger systems of covalent modification of enzyme proteins would be rendered ineffective by lack of glycogen substrate. The availability of SGP controls the amount and location of the cell's stores of glycogen and its discovery adds a new dimension to our knowledge of the economy of this important cellular fuel.